The Expression Profile Of Innate Immune Receptors TLRs And NLRs Families In Long-term Cultured Human Umbilical Cord Mesenchymal Stem Cells

AIM：To detect and analyze the expression of innate immune receptors TLRs andNLRs families in hUC-MSCs from long-term culture. And investigating thesubculture effect on hUC-MSCs features, including inflammatory response,immunomodulation, proliferation, differentiation and migration. Accumulating datafor the safety of the hUC-MSCs application in experiments and clinical treatments.METHODS: Colecting neonatal umbilical cord in aseptic condition. hUC-MSCswere isolated and purified with collagenase II digestion and adherence screeningmethods. And passage3and passage28hUC-MSCs were identified by flow cytometryand induced differentiation. According to Bustin S A’ standard, we design and test theprimers of33TLRs and NLRs family members. Using RT-qPCR to detect TLRs andNLRs expression in passage3and passage28hUC-MSCs.RESULTS: The cell phenotypes of passage3and passage28hUC-MSCs are both theCD29+/CD44+/CD105+/CD31-/CD34-/CD40-/CD45-/CD106-/HLA-DR, and twopassaged cells can be both induced into osteoblast and adipocyte. PCRproducts sequence analysis demonstrate that all of primers are specific for33TLRs and NLRs families members. TLRs family members are all expressedin hUC-MSCs, TLR1, TLR3and TLR6have high mRNA expression, TLR2,TLR4, TLR5and TLR10have middle mRNA expressioin, TLR7, TLR8andTLR9have low mRNA expression. The passage3hUC-MSCs compare withpassage28hUC-MSCs, TLR2and TLR3mRNA expression are increasing,TLR7mRNA expression have hardly changed, the rest members of TLRsmRNA expression are decreasing, and the difference of TLR3mRNAexpression has statistical significance (P<0.05). NLRs family members are allexpressed in hUC-MSCs, the NOD1, NLRC4, NLRC5, NLRP1, NLRP3, NLRP10,NAIP, NLRX1and APAF1mRNA are highly expressed, the rest members of NLRsmRNA are lowly expressed. The passage3hUC-MSCs compare with passage28hUC-MSCs, NLRP10mRNA are increasing, NLRC5and NLRX1mRNA have hardly changed, the rest members of NLRs mRNA expression aredecreasing, and the difference of NLRP1mRNA expression has statisticalsignificance (P<0.05).CONCLUSION: Experiment results show that cells still possessed the basicphenotype of MSCs after hUC-MSCs are cultured for28generations under normalcondition, but its abilities of proliferation and differentiation decrease. All33genes ofTLRs and NLRs family were expressed; the expression of genes from differentmembers has a little big difference. In the process of subculture hUC-MSCs, theexpressions of most genes from TLRs and NLRs family toward to downtrend, parts ofgenes’ expression are increased, decreased or no change, suggesting the effects ofsubculture on hUC-MSCs expressing TLRs and NLRs family are pleiotropic. Dueto different TLRs and NLRs family members and its signaling pathways have closerelationships with cells proliferation and differentiation, inflammatory reaction andimmune regulation, which may reflect the effects of long time culture on functionsrelated with hUC-MSCs. These results will provide the investigated targets forfunction interfere of hUC-MSCs, and give direction for improving the quality ofcultured cells and strengthening the cell funciton. These things will contribute forhUC-MSCs application in tissue engineering and clinical transplantation treatments.